Horizontal transfers and gene losses in the phospholipid pathway of bartonella reveal clues about early ecological niches.

1Department of Biological Sciences, University at Buffalo, State University of New York qiyunzhu@buffalo.edu kd52@buffalo.edu.

2Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Foothills Research Campus, Fort Collins, Colorado.

3EcoHealth Alliance, New York, New York.

4Department of Biological Sciences, University at Buffalo, State University of New York Graduate Program of Evolution, Ecology, and Behavior, University at Buffalo, State University of New York qiyunzhu@buffalo.edu kd52@buffalo.edu.

Abstract

Bartonellae are mammalian pathogens vectored by blood-feeding arthropods. Although of increasing medical importance, little is known about their ecological past, and host associations are underexplored. Previous studies suggest an influence of horizontal gene transfers in ecological niche colonization by acquisition of host pathogenicity genes. We here expand these analyses to metabolic pathways of 28 Bartonella genomes, and experimentally explore the distribution of bartonellae in 21 species of blood-feeding arthropods. Across genomes, repeated gene losses and horizontal gains in the phospholipid pathway were found. The evolutionary timing of these patterns suggests functional consequences likely leading to an early intracellular lifestyle for stem bartonellae. Comparative phylogenomic analyses discover three independent lineage-specific reacquisitions of a core metabolic gene-NAD(P)H-dependent glycerol-3-phosphate dehydrogenase (gpsA)-from Gammaproteobacteria and Epsilonproteobacteria. Transferred genes are significantly closely related to invertebrate Arsenophonus-, and Serratia-like endosymbionts, and mammalian Helicobacter-like pathogens, supporting a cellular association with arthropods and mammals at the base of extant Bartonella spp. Our studies suggest that the horizontal reacquisitions had a key impact on bartonellae lineage specific ecological and functional evolution.

Role of GpsA in Bartonella phospholipid biosynthesis. Part of the alphaproteobacterial phospholipid biosynthesis pathway is illustrated based on Cronan (2003), Pereto et al. (2004), Spoering et al. (2006), Yeh et al. (2008), and the KEGG pathway entry bhe00564 (glycerophospholipid metabolism in B. henselae). The illustration highlights the three possible paths of obtaining G3P. The dashed lines represent the reactions affected by ancient gene losses; and the bold line represents the reaction affected by one ancient gene loss followed by three independent horizontal regains in the evolutionary history of Bartonella.

Losses and gains of gpsA and metabolically related genes in the evolutionary history of Bartonella. Schematically illustrated relationships of Bartonella lineages based on phylogenomic and phylogenetic analyses of the 28 Bartonella species (supplementary fig. S1, Supplementary Material online). Topology is congruent with a recent phylogenomic study (Guy et al. 2013). Major monophyletic lineages (table 1) were collapsed into triangles. Branch lengths are not drawn to scale. The presence and origin of gpsA is indicated to the right of corresponding lineages. Horizontally acquired genes are indicated by gray boxes, whereas vertically inherited genes are indicated by white boxes. HGT events are represented by incoming arrows, with the putative donor groups (if identifiable) labeled. Gene loss events are represented by outgoing arrows and boxes with dashed outlines. Phylogenetic positions of losses and gains are indicated by circles.

Phylogenies of different versions of gpsA. Trees were reconstructed using Bayesian inference as implemented in MrBayes. Node labels (x/y) represent Bayesian posterior probabilities (x%) computed in MrBayes and ML bootstrap support values (y% out of 1,000 replicates) computed in RAxML. Asterisks (*) indicate 100% support. Bartonella clades are denoted in bold font. (A) Vertical inheritance history of gpsA (Rh) in Rhizobiales. Families Bartonellaceae, Brucellaceae, Phyllobacteriaceae, and Rhizobiaceae are placed as ingroups and the other Rhizobiales organisms as outgroups, according to Gupta and Mok (2007). (B) Horizontal transfer of gpsA (He) from Helicobacter to L1 and L2 Bartonella (including an experimentally verified deer ked sample). The tree is rooted at the common ancestor of Helicobacteraceae and Campylobacteraceae, according to Gupta (2006). (C) Horizontal transfers of gpsA (Ar) from Arsenophonus-like bacteria to L4 Bartonella, and that of gpsA (Se) from Serratia to B. australis. The tree includes recipient Bartonella species, representative Enterobacteriales groups, and two Arsenophonus-positive bat fly samples sequenced in this study. It is rooted to Vibrionales according to Williams et al. (2010). Monophyletic groups are collapsed in triangles with nodal support values labeled to the right. Long branches are truncated and indicated by two slashes (//). (D) Posttransfer evolutionary history of Arsenophonus-derived gpsA (Ar) in L4 Bartonella. This is an expansion of the L4 Bartonella clade in (C). Experimentally verified insect samples are indicated by the insect names. Nodal support values of derived clades are omitted.

Genomic context of gpsA (He) in Bartonella and other bacterial groups. Genes are represented by boxes. ORFs annotated as hypothetical genes are either indicated by “?,” or by single letters (e.g., “M” and “X,” see below). Lengths of genes and intergenic regions are not drawn to scale. “X” represents an ORF that is disrupted by the insertion of gpsA (He). “M” represents a multicopy ORF that exists only in B. bacilliformis and B. australis genomes.

Comparison of protein sequences of different GpsA versions. Alignment of full-length GpsA protein sequences to Coxiella burnettii. Nucleotide positions are shaded by similarity from low (light) to high (dark) on a grayscale. GpsA proteins are aligned in pairs with a representative sequence from the donor group and its Bartonella counterpart. Functional sites and motifs are boxed, as recorded in UniProtKB. Significantly positively selected sites predicted by BEB are indicated by solid triangles.